Krebs henseleit buffer

Adult Sprague Dawley rats (n = 8 male: 8 weeks, 265 g; and n = 8 female: 12 weeks, 255 g) were euthanized and hearts were removed and collected in Krebs-Henseleit buffer (Sigma, St. Louis, MO, USA). All procedures were performed with approval from Clemson University’s Institutional Animal Care and Use Committee (protocol AUP 2019-048). Ventricles were minced and digested to isolate CFs according to previously reported protocols [25 (link),26 (link)]. Liberase TM (Roche, Indianapolis, IN, USA) was used in each of the six successive enzymatic digestions at 37 °C. Supernatants from each digestion were collected and centrifuged at 300× g and 4 °C and resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA), 100 U/mL penicillin G, 100 µg/mL streptomycin, and 1 ng/mL amphotericin B (all Sigma). Following isolation, cells were plated in T-25 culture flasks and incubated at 37 °C and 5% CO₂ for 4 h after which media was changed and, thereafter, was changed every 72 h until the serum starvation.

Murine primary hepatocytes were isolated as described earlier (Malhi et al., 2006 (link); Dasgupta et al., 2020 (link)). Briefly, a 2-step collagenase perfusion technique was performed. After in situ perfusion of the liver with a calcium-free HBSS medium (Corning, Corning, NY, United States) followed by a collagenase D (Roche, Indianapolis, IN, United States) solution in HBSS medium with calcium and magnesium (Corning, Corning, NY, United States), livers were excised and minced in Krebs-Henseleit buffer (Sigma-Aldrich, St. Louis, MO, United States). Dispersed cell suspensions were purified by Percoll solution (Sigma). The hepatocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) in collagen coated plates, prepared with Collagen coating solution (Sigma-Aldrich, St. Louis, MO, United States). Approximately, 2.5×10⁶ cells were plated in 10 cm dishes, 1.5×10⁶ cells were plated in 60 mm dishes, and 750,000 cells per well in 6-wells plates.

To determine the time-dependent OAT1-mediated intracellular uptake of IS, mature monolayers of ciPTECs-OAT1 were incubated with IS (25, 50, 100, and 500 μM prepared in Krebs–Henseleit buffer (Sigma-Aldrich, Zwijndrecht, The Netherlands) supplemented with HEPES (10 mM, Sigma-Aldrich, Zwijndrecht, The Netherlands, pH 7.4) for variable periods of time. Uptake was stopped by washing one time with ice-cold HBSS (Life Technologies Europe BV, Roskilde, Denmark), and then, the cells were lysed by 100 µL 0.1M NaOH for 10 min at room temperature and under mild shaking. The intracellular IS concentration was determined in the cell lysate by LC-MS/MS, as described below.

Each muscle was carefully dissected into longitudinal strips from tendon to tendon using a 27-gauge needle. Following dissection, the gastrocnemius muscle was separated into red and white muscle and snap-frozen in liquid nitrogen for subsequent measurement of citrate synthase activity. Immediately after their removal the longitudinal strips of the soleus and EDL were incubated in an oxygenated organ bath (37 °C pre-gassed at 95% O₂ −5% CO₂). Each longitudinal strip was placed in an individual chamber of Krebs-Henseleit buffer (Sigma-Aldrich, St. Louis, MO, USA) with 0.1 µg/mL of adiponectin (Sigma-Aldrich, St. Louis, MO, USA) or the contralateral hind limb muscle exposed to Vehicle for 30 min. Muscle strips were taken from opposing limbs from each animal to ensure that alternate muscles were exposed to the treatment. Following incubation, the muscle samples were snap frozen in liquid nitrogen and stored at −80 °C for further analyses.

Discarded human pancreases were made available to the clinical team on two occasions. The same protocol was used as described for the porcine pancreases. Each pancreas was stored on ice before being subjected to 5 hours of HMP with in-house Belzer-UW solution, followed by 2 hours of reperfusion with warm oxygenated Krebs–Henseleit buffer (Sigma-Aldrich, UK). As with the porcine pancreases, the microdialysis probes were removed between the HMP and the reperfusion stages. Human pancreas (ii) was donated by a 62 year old man. The organ was offered for transplantation but was refused on the basis of donor age and the presence of atherosclerotic plaques. The pancreas was stored on ice for 25 hours before HMP commenced. In the case of human pancreas (iv), no information was available about the donor. The organ was offered for transplantation and was matched with a recipient, but the recipient was not fit for surgery and by that time the CIT was too long to match the organ to another recipient. The pancreas was stored on ice for 57 hours before HMP commenced. Fig. 1C shows a microdialysis probe positioned in a human pancreas during HMP.